BioMed Central Page 1 of 5 (page number not for citation purposes) Journal of Negative Results in BioMedicine Open Access Research Association study with Wegener granulomatosis of the human phospholipase C γ 2 gene Peter Jagiello* †1 , Stefan Wieczorek †1 , Philipp Yu 2 , Elena Csernok 3 , Wolfgang L Gross 3 and Joerg T Epplen 1 Address: 1 Department of Human Genetics, Ruhr-University Bochum Germany, 2 Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Germany and 3 Department of Rheumatology, University Hospital Luebeck and Rheumaklinik Bad Bramstedt, Germany Email: Peter Jagiello* - peter.jagiello@rub.de; Stefan Wieczorek - stefan.wieczorek@rub.de; Philipp Yu - philipp.yu@lrz.tu-muenchen.de; Elena Csernok - csernok@rheuma-zentrum.de; Wolfgang L Gross - gross@rheuma-zentrum.de; Joerg T Epplen - joerg.t.epplen@rub.de * Corresponding author †Equal contributors Abstract Background: Wegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, e.g. with α (1)-antitrypsin, proteinase 3 and with the HLA-DPB1 locus. A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseC γ 2 (PLC γ -2) gene, corresponding to human PLC γ -2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2 (HRL1 and 2, respectively) and exon 27 of the PLCγ-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLC γ -2 gene. Results: Although a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort. Conclusion: This report does not reveal any hints for an involvement of the PLC γ -2 gene in the pathogenesis of WG in our case-control study. Background Wegener granulomatosis (WG) is a systemic inflamma- tory disease of unknown aetiology characterized by gran- ulomata of the respiratory tract and systemic necrotizing vasculitis [1]. There is a strong and specific association with presence of anti-neutrophil cytoplasmatic antibodies to a defined target antigen, proteinase 3 (PR3-ANCA), which is present within primary azurophil granules of neutrophils (PMN) and lysozymes of monocytes [2]. Upon cytokine priming of PMNs, this enzyme translo- cates to the cell surface, where PR3-ANCAs can interact with their antigens and activate PMNs [3]. It has been shown that PMNs from patients with active WG express- ing PR3 on their surfaces produce respiratory burst and Published: 09 February 2005 Journal of Negative Results in BioMedicine 2005, 4:1 doi:10.1186/1477-5751-4-1 Received: 07 October 2004 Accepted: 09 February 2005 This article is available from: http://www.jnrbm.com/content/4/1/1 © 2005 Jagiello et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Journal of Negative Results in BioMedicine 2005, 4:1 http://www.jnrbm.com/content/4/1/1 Page 2 of 5 (page number not for citation purposes) release proteolytic enzymes after activation with PR3- ANCA [4]. The consequence is a self-sustaining chronify- ing inflammatory process. WG appears as a multifactorial disease, and environmental influences still remain elu- sive. Several factors, such as bacterial infections, have been proposed as probable initiators of the disease [5]. It has been reported that chronic carrier status of Staphyloco- ccus aureus is a risk factor for disease exacerbation in WG [6]. Recently a strong association of WG with distinct HLA-DPB1 alleles or rather an extended haplotype, respectively, in the MHC class II region has been reported [7]. In addition, analyses of candidate genes revealed sev- eral associations, e.g. with α (1)-antitrypsin, and protein- ase 3 [[8] and [9]]. A primary candidate gene, PLC γ -2, was proposed on the basis of a novel animal model system. A mutation in the abnormal limb mutant 5 (ALI5) mouse [10] causes a phe- notype comparable to human autoimmunity disease like WG: inflammations, granulomatosis, affected organs (lung, kidney with glomerulonephritis, eye and skin) and ANCAs were detected in the ALI5 mouse initially. Yet, this result has to be confirmed in further studies. The ALI5 mutation is located in the genomic region coding for the hydrophobic ridge loop 3. In mouse PLC γ -2 mutation leads to acute and chronic inflammations with a pheno- type comparable to WG. Therefore, human PLC γ -2 is a good candidate gene for seeking predisposing genetic fac- tors for WG. The human gene is a member of the PLC fam- ily comprising 12 closely related molecules involved in signal transduction from numerous receptors [11]. The protein is activated by cytoplasmatic tyrosine kinases (Lyn, Syk, and Btk) which are induced by engagement of B-cell receptors. In turn, the PLCγ-2 activation leads to the generation of diacylglycerol (DAG) and inositol 1,4,5-tri- sphosphat (IP3). While DAG activates protein kinase C (PKC, [12]), IP3 mediates Ca 2+ mobilization, which is required for activation of B-cells [13]. PLCγ-2 itself is expressed mainly in B-cell, hematopoetic cells, macro- phages, granulocytes, testis, sperm, skin and brain [14]. The protein consists of two catalytic domains, which are separated by two SH2 and one SH3 domains [15]. It was established that PLCγ-2 mediates the coupling of G-pro- tein-coupled receptors (GPCRs) and Ca 2+ entry in cell lines [16]. The human PLC γ -2 gene is localized on chro- mosome 16 (16q24.1) spanning 179 kb of genomic DNA. The 4.2 kb mRNA consists of 33 Exon coding for a M r 140,000 protein with 1265 amino acids [17]. PLC γ -2 is highly polymorphic [18]. Here, we report on an indirect association screen by mic- rosatellite analysis and pooling of DNA in a case-control study design. Furthermore, we screened exons 11, 12 and 13, partly coding for the hydrophobic ridge loop 1 and 2 of PLCγ-2 protein, by single stranded conformation poly- morphism (SSCP), sequencing and the PCR/RFLP method. As the ALI5 mutation is located in the region cor- responding to the human exon 27, this exon was also screened by SSCP. Results Analyses of microsatellites In the present study we analysed 4 microsatellites intra- or juxta-genic of the PLC γ -2 gene with pooled DNAs from WG patients compared with those from controls (table 1; figure 1). All markers exhibited at least 3 alleles and did not show any intra-subgroup differences. The microsatel- lite analyses did not reveal significantly different allele distributions between WG patient and control pools (fig- ure 1). SSCP, sequencing and PCR/RFLP analyses SSCP analysis was chosen to identify mutations or poly- morphisms, respectively, in exons 11, 12, 13 and 27 of the PLC γ -2 gene. DNA's of patients and controls with altered migration behaviour were sequenced. Identified varia- tions were genotyped individually by SSCP and/or PCR/ RFLP in individual patient and control samples (see table 2). Exon 12 revealed a low frequent SNP (1122G>A), which represents the first base pair of the exon. In exon 13 two previously identified SNPs (1293T>C and 1296T>C) were detected [18]. Both SNPs showed similar frequencies as reported [18]. All variations were found not signifi- cantly different in WG patients compared to the control Table 1: Primer sequences and information about microsatellites used in study Primer sequence No. sense 1 antisense *nucleotide marker 1 CGCACATGTATCCAGAACT AGAGGTGGACCCATGCTTA *di (AT) 2 CAAAGAAGATAAGGGCAGGC CCTAGGCGACTCAGTGAGACT *tetra (TTTA) 3 AGGAGTTCGAGAAGAGCCTG TGCCACTACACCCAGATGAT *di (AC) 4 TGATCTGTGTCTGGGCTTTC AGTTGTGACCCTAACATTGCA *di (AC) 1 The fluorescence labelled tail (5-Fam-CATCGCTGATTCGCACAT) was added to the 5'end of each sense primer Journal of Negative Results in BioMedicine 2005, 4:1 http://www.jnrbm.com/content/4/1/1 Page 3 of 5 (page number not for citation purposes) group, and they were not associated with amino acid sub- stitutions. In one patient our analyses revealed a single base substitution (3030G>A) in exon 27. Discussion As recently reported a mutation in the PLC γ -2 gene in the ALI5 mouse leads to acute and chronic inflammation and granulomatosis. In addition, the ALI5 mice show similar symptoms as WG patients. For this reason screening of PLC γ -2 appears as a logical consequence in seeking candi- date genes for WG. In the present study PLC γ -2 was ana- lysed by an indirect microsatellite approach using pooled DNA from WG patients with a defined PR3-ANCA + status and a matched control cohort as reported before [7]. Fur- thermore, exons partly representing the catalytic domains of the PLCγ-2 protein, HRL1, 2 and 3, were analysed by SSCP, sequencing and by the PCR/RFLP method. We did not find any hints of an involvement of the PLC γ -2 gene in the pathophysiology of WG. Hence, we exclude at this time that the PLC γ -2 gene predisposes for WG in our cohort. Our study revealed 4 single base substitutions, 2 of which were reported before [18]. A further silent and low fre- quent SNP was detected in exon 12 which did not differ significantly between patient and control cohorts after PCR/RFLP analyses. As this SNP is the first basepair (bp) after the 3'-splice site one might hypothesise an influence on splicing but to present knowledge this SNP does not change a consensus sequence required for splicing. The ALI5 mutation is located in the HRL3 domain partly corresponding to the human exon 27 of PLC γ -2. Our analysis revealed a SNP (G>A) at position 3030 in one WG patient. Schematic representation of the human PLCγ-2 gene with relative localization of exons and investigated microsatellitesFigure 1 Schematic representation of the human PLCγ-2 gene with relative localization of exons and investigated microsatellites. P val- ues were generated by contingency tables (for details see "materials and methods"). Vertical lines: exons; No1-6: investigated microsatellites 1-6; Ex: exon. Table 2: Summary of found variations in the human PLC γ -2 gene in exons 11, 12, 13 and 27 variation frequency of alleles exon bp 1 codon WG patients controls P value restriction enzymes 11 12 1122G>A T329T 3/262 3/162 0.55 BanI 13 1293T>C 2 F382F 7/350 4/324 0.68 PfIFI 1296T>C 2 D383D 143/262 96/186 0.78 TaqI 27 3030G>A T961T 1/166 3 0/180 3 0.51 - 1 Numbering according to BC007565 (UCSC); 2 Previously reported SNPs; 3 Frequencies determined by SSCP analyses Journal of Negative Results in BioMedicine 2005, 4:1 http://www.jnrbm.com/content/4/1/1 Page 4 of 5 (page number not for citation purposes) The indirect microsatellite based approach did not reveal any association of PLC γ -2 with WG. Altogether 4 micros- atellites spread in the PLC γ -2 gene were analysed. The approach using pooled DNA and ad hoc designed markers intragenic or in the immediate vicinity of a distinct gene has proven to be a reliable and efficient method to detected predisposing loci in WG before [7]. Here, none of the markers did show a significant allele distribution between the patient and control group. Conclusion In conclusion, our analysis of the human PLC γ -2 gene did not reveal an association of PLC γ -2 with WG. In contrast to ALI5 mice, where a single mutation leads to distinct symptoms of inflammatory autoimmunity, human WG depends on a more complex genetic background. Further analysis of all exons of PLC γ -2 might yield an association with WG but our microsatellite analysis strongly suggests that predisposition for WG is not due to variations in the PLC γ -2 gene. Material and Methods Patients and controls 175 well-characterized patients of German origin with a clinical diagnosis of WG and a defined PR3-ANCA + status were included in present study. Diagnosis of WG was established according international standards [[19] and [20]]. All patients were biopsy-proven. Biopsies were seen in German reference centre for vasculitis (Department of Pathology, University of Schleswig-Holstein Campus Lue- beck, Germany) by 2 different observers. A group of 165 healthy individuals of German origin were used as con- trols. All persons gave informed consent. Microsatellite analysis Pooling of DNA was performed as reported before [7]. Patient (n = 150) and healthy control (n = 100) individu- als from the abovementioned groups were divided into 3 and 2 sub-pools, respectively, containing 50 persons each. In this study 3 intragenic microsatellites as well as one in the immediate vicinity of the gene were included (table 1; see also UCSC Database, June 2002 Freeze; URL). Oligo- nucleotides were designed by Primer Express 2.0 software (ABI) adjusted to an annealing temperature of 55°C. For PCR we used the 'tailed primer PCR' as described before [7]. For amplification three oligonucleotides were used: 1. tailed sense primer (tailed F), 2. anti-sense primer and 3. labeled primer (labeled F) corresponding to the 5'- tail sequence of tailed F. PCR conditions were as follows: 1 × PCR buffer (Qiagen), 1.5 pmol labeled F, 0.2 mM each dNTP, 3 mM MgCl 2 , 0.2 pmol tailed F, 1.5 pmol reverse primer, 0.25 U Qiagen Hot Start Taq (Qiagen) and 50 ng DNA. Electrophoreses were run using ABI standard protocols. Raw data were analyzed by the Genotyper software (ABI) producing a marker-specific allele image profile (AIP, see [21] and [7]). AIP consists of series of peaks with different heights reflecting the allele frequency within each ana- lyzed DNA pool. Statistics for comparisons of allele frequencies of patients and controls was performed as described before [[21] and [7]]. Case and control distributions were compared statis- tically by means of contingency tables. SSCP, sequencing and PCR/RFLP analyses Exons 11, 12, 13 and 27 were analysed by the SSCP method. PCR was performed using oligonucleotides reported before ([18], exon 27 corresponding to exon 26). Heat-denaturated fragments were then separated by poly- acrylamide gel electrophoresis under non-denaturating conditions yielding specific band patterns for each of the alleles. Results were visualized by autoradiography. Alle- les of representative probes were determined by direct DNA sequence analysis on a 377 ABI automatic sequencer (ABI). Afterwards, variations were genotyped individually by PCR/RFLP method with restriction enzymes specific for the respective change (for details see table 2). The varia- tion in exon 27 was individually genotyped by the SSCP method. Authors' contribution PJ and SW carried out the molecular genetic studies, per- formed the statistical analysis and drafted the manuscript. PY participated in study design and helped to draft the manuscript. EC and WLG provided the samples and per- formed diagnostics of the patient group. JTE conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. References 1. Lamprecht P, Gross WL: Wegener's granulomatosis. Herz 2004, 29:47-56. 2. Csernok E, Muller A, Gross WL: Immunopathology of ANCA- associated vasculitis. Intern Med 1999, 38:759-765. 3. Csernok E, Ernst M, Schmitt W, Bainton DF, Gross WL: Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo. Clin Exp Immunol 1994, 95:244-250. 4. van der Geld YM, Limburg PC, Kallenberg CG: Proteinase 3, Wegener's autoantigen: from gene to antigen. J Leukoc Biol 2001, 69:177-190. 5. Brons RH, Bakker HI, Van Wijk RT, Van Dijk NW, Muller Kobold AC, Limburg PC, Manson WL, Kallenberg CG, Tervaert JW: Staphyloco- ccal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener's granulomatosis? Clin Exp Immunol 2000, 119:566-573. 6. Popa ER, Tervaert JW: The relation between Staphylococcus aureus and Wegener's granulomatosis: current knowledge and future directions. Intern Med 2003, 42:771-780. 7. Jagiello P, Gencik M, Arning L, Wieczorek S, Kunstmann E, Csernok E, Gross WL, Epplen JT: New genomic region for Wegener's granulomatosis as revealed by an extended association screen with 202 apoptosis-related genes. Hum Genet 2004, 114:468-477. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Journal of Negative Results in BioMedicine 2005, 4:1 http://www.jnrbm.com/content/4/1/1 Page 5 of 5 (page number not for citation purposes) 8. Esnault VL, Testa A, Audrain M, Roge C, Hamidou M, Barrier JH, Ses- boue R, Martin JP, Lesavre P: Alpha 1-antitrypsin genetic poly- morphism in ANCA-positive systemic vasculitis. Kidney Int 1993, 43:1329-1332. 9. Gencik M, Meller S, Borgmann S, Fricke H: Proteinase 3 gene pol- ymorphisms and Wegener's granulomatosis. Kidney Int 2000, 58:2473-2477. 10. Yu P, Constien R, Dear N, Katan M, Hanke P, Bunney TD, Kunder S, Quintanilla-Martinez L, Huffstadt U, Schroeder A, Jones NP, Peters T, Fuchs H, Hrabe de Angelis M, Nehls M, Grosse J, Wabnitz P, Meyer TPH, Yasuda K, Schiemann M, Schneider-Fresenius C, Jagla W, Russ A, Popp A, Josephs M, Marquardt A, Laufs J, Schmittwolf C, Wagner H, Pfeffer K, Mudde GC: Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase Cγ2 that spe- cifically increases external Ca 2+ entry. Immunity in press. 11. Rhee SG: Regulation of phosphoinositide-specific phospholi- pase C. Annu Rev Biochem 2001, 70:281-312. 12. Rhee SG, Bae YS: Regulation of phosphoinositide-specific phos- pholipase C isozymes. J Biol Chem 1997, 272:15045-15048. 13. Kurosaki T, Maeda A, Ishiai M, Hashimoto A, Inabe K, Takata M: Reg- ulation of the phospholipase C-gamma2 pathway in B cells. Immunol Rev 2000, 176:19-29. 14. Marshall AJ, Niiro H, Yun TJ, Clark EA: Regulation of B-cell acti- vation and differentiation by the phosphatidylinositol 3- kinase and phospholipase Cgamma pathway. Immunol Rev 2000, 176:30-46. 15. Manning CM, Mathews WR, Fico LP, Thackeray JR: Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins. Genetics 2003, 164:433-442. 16. Patterson RL, van Rossum DB, Ford DL, Hurt KJ, Bae SS, Suh PG, Kurosaki T, Snyder SH, Gill DL: Phospholipase C-gamma is required for agonist-induced Ca2+ entry. Cell 2002, 111:529-541. 17. Emori Y, Homma Y, Sorimachi H, Kawasaki H, Nakanishi O, Suzuki K, Takenawa T: A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity. J Biol Chem 1989, 264:21885-21890. 18. Wang D, Boylin EC, Minegishi Y, Wen R, Smith CI, Ihle JN, Conley ME: Variations in the human phospholipase Cgamma2 gene in patients with B-cell defects of unknown etiology. Immunoge- netics 2001, 53:550-556. 19. Leavitt RY, Fauci AS, Bloch DA, Michel BA, Hunder GG, Arend WP, Calabrese LH, Fries JF, Lie JT, Lightfoot RW: The American Col- lege of Rheumatology 1990 criteria for the classification of Wegener's granulomatosis. Arthritis Rheum 1990, 33:1101-1107. 20. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, Hagen EC, Hoffman GS, Hunder GG, Kallenberg CG: Nomencla- ture of systemic vasculitides: Proposal of an international consensus conference. Arthritis Rheum 1994, 37:187-192. 21. Goedde R, Sawcer S, Boehringer S, Miterski B, Sindern E, Haupts M, Schimrigk S, Compston A, Epplen JT: A genome screen for link- age disequilibrium in HLA-DRB1*15-positive Germans with multiple sclerosis based on 4666 microsatellite markers. Hum Genet 2002, 111:270-277. . Central Page 1 of 5 (page number not for citation purposes) Journal of Negative Results in BioMedicine Open Access Research Association study with Wegener granulomatosis of the human phospholipase. gene in the pathogenesis of WG in our case-control study. Background Wegener granulomatosis (WG) is a systemic inflamma- tory disease of unknown aetiology characterized by gran- ulomata of the respiratory. the Genotyper software (ABI) producing a marker-specific allele image profile (AIP, see [21] and [7]). AIP consists of series of peaks with different heights reflecting the allele frequency within